Reduction of graft-versus-host disease by histone deacetylase inhibitor suberonylanilide hydroxamic acid is associated with modulation of inflammatory cytokine milieu and involves inhibition of STAT1
2006; Elsevier BV; Volume: 34; Issue: 6 Linguagem: Inglês
10.1016/j.exphem.2006.02.014
ISSN1873-2399
AutoresCorinna Leng, Margarethe Gries, Judy Ziegler, Anna Lokshin, Paolo Mascagni, Suzanne Lentzsch, Markus Y. Mapara,
Tópico(s)Immune Cell Function and Interaction
ResumoHistone deacetylase (HDAC) inhibitors reduce development of graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT). Administration of the HDAC inhibitor suberonylanilide hydroxamic acid (SAHA) resulted in a significantly reduced GVHD-dependent mortality following fully major histocompatibility complex–mismatched allogeneic BMT. However, SAHA treatment did not affect T-cell activation or T-cell expansion in vitro and in vivo. Therefore, we focused on the effects of SAHA treatment on cytokine production and intracellular signaling events in vitro and in vivo following GVHD induction. Cultivation in the presence of SAHA broadly inhibited lipopolysaccharide (LPS) and alloantigen-induced cytokine/chemokine production in vitro and led also to a significant decrease in interferon-γ and tumor necrosis factor-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and alloactivation in vitro. Induction of GVHD led to a rapid phosphorylation of STAT 1 in the liver and spleen, which was markedly reduced by SAHA treatment. In conclusion, GVHD is associated with a marked induction of phosphorylation of STAT1 in the liver and spleen, and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of phosphorylated STAT1 and blunting proinflammatory cytokine production during the initiation phase of GVHD. Histone deacetylase (HDAC) inhibitors reduce development of graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT). Administration of the HDAC inhibitor suberonylanilide hydroxamic acid (SAHA) resulted in a significantly reduced GVHD-dependent mortality following fully major histocompatibility complex–mismatched allogeneic BMT. However, SAHA treatment did not affect T-cell activation or T-cell expansion in vitro and in vivo. Therefore, we focused on the effects of SAHA treatment on cytokine production and intracellular signaling events in vitro and in vivo following GVHD induction. Cultivation in the presence of SAHA broadly inhibited lipopolysaccharide (LPS) and alloantigen-induced cytokine/chemokine production in vitro and led also to a significant decrease in interferon-γ and tumor necrosis factor-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and alloactivation in vitro. Induction of GVHD led to a rapid phosphorylation of STAT 1 in the liver and spleen, which was markedly reduced by SAHA treatment. In conclusion, GVHD is associated with a marked induction of phosphorylation of STAT1 in the liver and spleen, and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of phosphorylated STAT1 and blunting proinflammatory cytokine production during the initiation phase of GVHD. The widespread application of allogeneic stem cell transplantation is limited due to considerable treatment-related morbidity and mortality, with graft-versus-host disease (GVHD) being the most serious clinical problem [1Gratwohl A. Hermans J. Apperley J. et al.Acute graft-versus-host disease: grade and outcome in patients with chronic myelogenous leukemia.Blood. 1995; 86: 813-818PubMed Google Scholar] even after the introduction of nonmyeloablative conditioning regimens [2McSweeney P.A. Niederwieser D. 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However, there are substantial differences between humans and mice with regard to tolerance induction across major histocompatibility complex (MHC) barriers, GVHD sensitivity, or establishment of stable mixed chimerism following allogeneic BMT, which warrant caution when extrapolating results from mouse BMT models to the clinical situation. The pathogenesis of GVHD is influenced by the immunogenetic [6Beatty P.G. Anasetti C. Hansen J.A. et al.Marrow transplantation from unrelated donors for treatment of hematologic malignancies: effect of mismatching for one HLA locus.Blood. 1993; 81: 249-253PubMed Google Scholar, 7Goulmy E. Schipper R. 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Moller A. et al.Increased serum levels of tumor necrosis factor precede major complications of bone marrow transplantation.Blood. 1990; 75: 1011-1016PubMed Google Scholar]. Intestinal damage and subsequent translocation of bacterial products including lipopolysaccharide (LPS) into the circulation have been shown to be central to the subsequent pathogenic events occurring during development of GVHD [11Hill G.R. Ferrara J.L. The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation.Blood. 2000; 95: 2754-2759PubMed Google Scholar]. In addition to alloactivation of donor T cells by host APCs, LPS appears to be a particularly important cofactor in the initiation phase of GVHD as shown by the effect of TLR4 mutation on the development of GVHD [12Cooke K.R. Hill G.R. Gerbitz A. et al.Hyporesponsiveness of donor cells to lipopolysaccharide stimulation reduces the severity of experimental idiopathic pneumonia syndrome: potential role for a gut-lung axis of inflammation.J Immunol. 2000; 165: 6612-6619PubMed Google Scholar], which is corroborated by preclinical and clinical data on the beneficial effect of gnotobiotic conditions on the development of GVHD [13Van Bekkum D.W. Knaan S. Role of bacterial microflora in development of intestinal lesions from graft-versus-host reaction.J Natl Cancer Inst. 1977; 58: 787-790Crossref PubMed Scopus (104) Google Scholar, 14Beelen D.W. Haralambie E. Brandt H. et al.Evidence that sustained growth suppression of intestinal anaerobic bacteria reduces the risk of acute graft-versus-host disease after sibling marrow transplantation.Blood. 1992; 80: 2668-2676PubMed Google Scholar]. Furthermore, recent data suggest that Peyer's patches appear to be the prime site where many of the relevant pathogenic factors coalesce [15Murai M. Yoneyama H. Ezaki T. et al.Peyer's patch is the essential site in initiating murine acute and lethal graft-versus-host reaction.Nat Immun. 2003; 4: 154-160Crossref Scopus (246) Google Scholar, 16Beilhack A. Schulz S. Baker J. et al.In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets.Blood. 2005; 106: 1113-1122Crossref PubMed Scopus (270) Google Scholar]. Interfering with the proinflammatory response rather than using globally immunosuppressive agents is a potentially attractive approach for inhibiting the development of GVHD while maintaining a GVL response. Considerable evidence has accumulated over the years showing that epigenetic mechanisms are involved in the regulation of chromatin structure and subsequent repression of gene expression. Histone acetylation has been associated with enhanced gene transcription, whereas deacetylation leads to chromatin compaction and repressed transcription [17Marks P.A. Richon V.M. Breslow R. Rifkind R.A. Histone deacetylase inhibitors as new cancer drugs.Curr Opin Oncol. 2001; 13: 477-483Crossref PubMed Scopus (509) Google Scholar, 18Yoshida M. Furumai R. Nishiyama M. Komatsu Y. Nishino N. Horinouchi S. Histone deacetylase as a new target for cancer chemotherapy.Cancer Chemother Pharmacol. 2001; 48: S20-S26Crossref PubMed Scopus (221) Google Scholar]. However, acetylation of nonhistone proteins is an additional mechanism how histone deacetylases (HDACs) regulate transcription. In line with this concept it has been demonstrated that acetylation of STAT3 is critical for its dimerization, cytokine-induced DNA binding, and transcription [19Yuan Z.L. Guan Y.J. Chatterjee D. Chin Y.E. Stat3 dimerization regulated by reversible acetylation of a single lysine residue.Science. 2005; 307: 269-273Crossref PubMed Scopus (612) Google Scholar] or that acetylation of nuclear factor kappaB (NF-κB) prevents interaction with its inhibitor IκB and leads to prolonged gene transcription [20Chen L. Fischle W. Verdin E. Greene W.C. Duration of nuclear NF-kappaB action regulated by reversible acetylation.Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1040) Google Scholar]. Furthermore, HDACs have been also shown to be involved in the regulation of inflammatory responses and histone deacetylase 3 is involved in repression of LPS-induced tumor necrosis factor (TNF) gene expression [21Mahlknecht U. Will J. Varin A. Hoelzer D. Herbein G. Histone deacetylase 3, a class I histone deacetylase, suppresses MAPK11-mediated activating transcription factor-2 activation and represses TNF gene expression.J Immunol. 2004; 173: 3979-3990Crossref PubMed Scopus (68) Google Scholar]. A novel family of compounds that inhibit histone deacetylases have been shown to positively and negatively regulate the expression of a number of target genes. HDAC inhibitors induce apoptosis, growth inhibition, or cell cycle arrest in tumor cells. Thus, HDAC inhibitors have been introduced in the treatment of various neoplasms [22Kelly W.K. Richon V.M. O'Connor O. et al.Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously.Clin Cancer Res. 2003; 9: 3578-3588PubMed Google Scholar, 23Sandor V. Bakke S. Robey R.W. et al.Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in patients with refractory neoplasms.Clin Cancer Res. 2002; 8: 718-728PubMed Google Scholar]. In addition, HDAC inhibitors exert immunomodulatory effects and significantly reduce the extent of glomerulonephritis-mediated renal disease observed in the MLR-lpr/lpr mouse model, suggesting that these compounds may have benefit in the treatment of patients with systemic lupus erythematosus [24Mishra N. Reilly C.M. Brown D.R. Ruiz P. Gilkeson G.S. Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse.J Clin Invest. 2003; 111: 539-552Crossref PubMed Scopus (357) Google Scholar]. Furthermore, HDAC inhibitors have an immunosuppressive effect on T cells as indicated by the inhibitory effect on activation-induced expression of CD25 and CD40-ligand [25Skov S. Rieneck K. Bovin L.F. et al.Histone deacetylase inhibitors: a new class of immunosuppressors targeting a novel signal pathway essential for CD154 expression.Blood. 2003; 101: 1430-1438Crossref PubMed Scopus (77) Google Scholar] in vitro. Recently, it was shown that the HDAC inhibitor suberonylanilide hydroxamic acid (SAHA) suppresses activated cytokine genes and can reduce LPS-induced TNF-α, interleukin (IL)-6, interferon-γ (IFN-γ), and IL-1β levels, concanavalin A-dependent hepatic cellular injury [26Leoni F. Zaliani A. Bertolini G. et al.The antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid exhibits antiinflammatory properties via suppression of cytokines.Proc Natl Acad Sci U S A. 2002; 99: 2995-3000Crossref PubMed Scopus (435) Google Scholar], and development of GVHD [27Reddy P. Maeda Y. Hotary K. et al.Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect.Proc Natl Acad Sci U S A. 2004; 101: 3921-3926Crossref PubMed Scopus (244) Google Scholar]. We sought to elucidate the mechanisms by which SAHA treatment interferes with the development of GVHD. Given the anti-inflammatory properties of SAHA, we hypothesized that HDAC inhibitor–dependent mitigation of GVHD might involve inhibition of major inflammatory signaling pathways in GVHD target organs with particular focus on LPS- and alloantigen-mediated inflammatory signaling events. Indeed, we were able to confirm and extend the results by Reddy et al. [27Reddy P. Maeda Y. Hotary K. et al.Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect.Proc Natl Acad Sci U S A. 2004; 101: 3921-3926Crossref PubMed Scopus (244) Google Scholar] in a fully MHC-mismatched model of GVHD, details of which are provided here. Treatment with SAHA led to delayed development of GVHD and was associated with significant inhibition of the systemic inflammatory reaction occurring shortly after myeloablative conditioning and BMT. This protective effect against GVHD was accompanied by modulation of the JAK-STAT pathway without impairing T-cell expansion and activation. Therefore, SAHA-dependent inhibition of GVHD is mediated primarily by attenuating the inflammatory milieu and might act by providing end-organ protection. Further studies are warranted to dissect the precise means by which HDAC inhibition interferes with signaling pathways in vivo. Furthermore, using molecular targeted therapy to block inflammatory signaling pathways such as those explored here may indeed be promising for the prophylaxis of GVHD and may help to improve treatment outcome following allogeneic transplantation. Female C57BL/6 (B6) and BALB/c mice were purchased from Charles River (Sulzfeld, Germany) and used after 8 weeks of age. All mice were housed in autoclaved microisolator environments and were provided with sterile water and irradiated food ad libitum. All manipulations were performed in a laminar flow hood. Recipient mice were lethally irradiated with 9.5 Gy at a dose rate of 1 Gy/min and were reconstituted within 4 hours with a single intravenous inoculum of either 1.5 × 107 allogeneic bone marrow cells ± spleen cells (1.5 × 107) or 5 × 106 syngeneic bone marrow cells. To avoid bias from cage-related effects, animals in different groups were randomized between cages. Animals received SAHA or vehicle by gavage from day −1 to day +10. Mixed lymphocyte reaction (MLR) assays were performed as previously described [28Mapara M.Y. Pelot M. Zhao G. Swenson K. Pearson D. Sykes M. Induction of stable long-term mixed hematopoietic chimerism following a cyclophosphamide-based non-myeloablative conditioning regimen leads to donor-specific in vitro and in vivo tolerance.Biol Blood Marrow Transplant. 2001; 7: 646-655Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar]. Briefly, stimulator cells were irradiated with 3000 rad using a 137Cs source. Four hundred thousand stimulator cells were cocultured with 4 × 105 responder cells in 96-well flatbottom plates for 3 to 4 days at 37°C before they were pulsed with 1 μCi of 3H-thymidine per well for the last 16 to 18 hours of culture. Cells were harvested upon glass filter mats using a Tomtec harvesting device (Hamden, CT, USA). 3H-thymidine incorporation was measured using a betaplate beta-counter. Spleen cells were studied by two-color or three-color flow cytometry using a FACScan cytometer (Becton Dickinson, Mountain View, CA, USA). Spleens were harvested and gently teased in bone marrow medium [Medium 199 (Mediatech), containing 2 μg/mL DNAse, 0.01 M HEPES buffer (Cambrex, Walkersville, ND, USA) and 4 μg/mL gentamycin] or ACK-lysing buffer (Cambrex), respectively. Single-cell suspensions were filtered through nylon mesh. For double- or triple-color staining, 106 cells were incubated in the presence of directly fluorescein-isothiocyanate (FITC)-, phycoerythrin (PE)-, or biotin (bio)-labeled monoclonal antibodies (mAbs) for 30 minutes at 4°C. Development of bio-labeled mAbs was performed by subsequent incubation with PE-conjugated avidin for 10 minutes. To reduce nonspecific binding of mAbs, 10 μL of 2.4G2 (anti-Fcγ-RII-receptor, CDw32) hybridoma supernatant was added to all tubes. The following antibodies were used for phenotyping: anti-CD4-FITC, anti-CD8β-FITC, anti-B220-FITC (all purchased from BD Biosciences Pharmingen, San Diego, CA, USA), anti-Mac-1-FITC (CalTag, San Francisco, CA, USA), KH-95-Bio (anti-H2-Kb). Nonreactive isotype control mAb (BD Biosciences Pharmingen) were used as negative controls. Exclusion of dead cells was performed by propidium iodide (PI) staining and live gating on PI-negative cells. Ten thousand events were collected and analyzed. For histone preparation, spleens were harvested followed by red blood cell lysis using ACK buffer. Thereafter, cells were washed twice with phosphate-buffered saline. Histones were isolated as described elsewhere [24Mishra N. Reilly C.M. Brown D.R. Ruiz P. Gilkeson G.S. Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse.J Clin Invest. 2003; 111: 539-552Crossref PubMed Scopus (357) Google Scholar]. Briefly, cells were pelleted (1–2 × 106) and resuspended in 1 mL ice-cold lysis buffer (10 mM TrisHCl [pH 6.5], 50 mM sodium bisulfite, 1% Triton X-100, 10 mM MgCl2, 8.6% sucrose). Nuclei were washed three times with 1 mL lysis buffer and once with Tris·ethylenediamine-tetraacetic acid (EDTA) solution (10 mM TrisHCl [pH 7.4], 13 mM EDTA). Nuclei were then pelleted and resuspended in 100 μL of ice-cold water. Samples were acidified with 0.2 mM sulfuric acid, vortexed thoroughly, and incubated for 1 hour. Thereafter, samples were centrifuged at 15,000g for 10 minutes at 4°C, and the protein in the supernatant was precipitated with 1 mL of acetone overnight at −20°C. Precipitated protein was collected by centrifugation at 15,000g for 5 minutes at 4°C (microcentrifuge), air-dried, and resuspended in 50 μL of water. Protein concentrations were quantified using a protein assay kit (Bio-Rad Laboratories, Munich, Germany). Cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond C super filters (Amersham, Arlington Heights, IL, USA). The blots were probed with the following antibodies: rabbit polyclonal antibodies against acetylated histone H3 and H4 (Upstate Biotechnology, Lake Placid, NY, USA) and immune complexes were detected using enhanced chemiluminescence (Amersham). Extraction of all other cellular proteins for immunoblotting was performed as previously described [29Lentzsch S. Gries M. Janz M. Bargou R. Dorken B. Mapara M.Y. Macrophage inflammatory protein 1-alpha (MIP-1 alpha) triggers migration and signaling cascades mediating survival and proliferation in multiple myeloma (MM) cells.Blood. 2003; 101: 3568-3573Crossref PubMed Scopus (189) Google Scholar]. Antibodies against p-STAT1, total STAT1, p-STAT3, STAT3, and glyceraldehyde-3-phosphate-dehydrogenase GAPDH were purchased from Cell Signaling (Beverly, MA, USA) and β-actin from Sigma-Aldrich (St. Louis, MO, USA). Densitometric analysis of scanned blots was done using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997–2005). Collection of supernatants and cell pellets from stimulated cell cultures was performed as follows: 5 × 106 BALB/c splenocytes were cultured alone or together with 5 × 106 irradiated (3000 rad) B6 splenocytes in 2 mL of MLR medium in 24-well plates. As indicated LPS was added at a final concentration of 100 ng/mL. All cultures were carried out with 0, 0.5, or 1 μM SAHA. Cells and supernatants were harvested after 4, 12, 24, and 48 hours of stimulation. The LabMap technology (Luminex Corp., Austin, TX, USA) combines the principle of a sandwich immunoassay with the fluorescent bead–based technology allowing individual and multiplex analysis of up to 100 different analytes in a single microtiter well [30Oliver K.G. Kettman J.R. Fulton R.J. Multiplexed analysis of human cytokines by use of the FlowMetrix system.Clin Chem. 1998; 44: 2057-2060PubMed Google Scholar, 31de Jager W. te Velthuis H. Prakken B.J. Kuis W. Rijkers G.T. Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.Clin Diagn Lab Immunol. 2003; 10: 133-139Crossref PubMed Scopus (498) Google Scholar]. The LabMap serum/supernatant assays were performed in 96-well microplate format according to a protocol provided by BioSource International (Camarillo, CA, USA). A filter-bottom, 96-well microplate (Millipore, Billerica, MA, USA) was blocked for 10 minutes with phosphate-buffered saline/bovine serum albumin. To generate a standard curve, fivefold dilutions of appropriate standards were prepared in serum or culture medium diluent. Standards, mouse sera, and culture supernatants were pipetted at 50 μL/well in duplicate and mixed with 50 μL of the bead mixture. The microplate was incubated for 1 hour at room temperature on a microtiter shaker. Wells were then washed three times with washing buffer using a vacuum manifold. PE-conjugated secondary antibody was added to the appropriate wells and the wells were incubated for 45 minutes in the dark with constant shaking. Wells were washed twice, assay buffer was added to each well, and samples were analyzed using the Bio-Plex suspension array system (Bio-Rad Laboratories, Hercules, CA, USA). Serum samples were analyzed using the mouse cytokine/chemokine Lincoplex assay (Lincoresearch, St. Charles, MO, USA) and supernatants using the mouse cytokine 20-plex from Biosource (Camarillo, CA, USA). Analysis of experimental data was performed using five parametric-curve fitting. As we were interested in studying the mechanisms how SAHA affects the development of GVHD, we aimed at confirming the results published by Reddy and colleagues [27Reddy P. Maeda Y. Hotary K. et al.Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect.Proc Natl Acad Sci U S A. 2004; 101: 3921-3926Crossref PubMed Scopus (244) Google Scholar]. For this purpose GVHD was induced in fully MHC-mismatched BALB/c → B6 strain combinations. Mice received 35 mg/kg SAHA orally from day −1 through day +10 post-BMT. Figure 1A shows pooled results from two experiments. SAHA treatment led to prolonged survival. Median survival time for vehicle- and SAHA-treated mice were 7.5 days and 38 days, respectively. Landmark analysis revealed that SAHA significantly reduced posttransplant mortality at days +10 and + 37 post-BMT. Thus, at days +10 or +37 post-BMT survival for vehicle-treated or SAHA-treated mice was 33% vs 86% and 8% vs 57%, respectively (chi-square test, p = 0.027 and p = 0.02, respectively). In addition to these confirmatory studies we found further evidence to substantiate our hypothesis that SAHA interferes with early events during induction of GVHD. Thus in a separate set of experiments SAHA treatment was delayed until day +10 post-BMT and continued until day +60. Using this treatment schedule SAHA did not influence the development of GVHD (data not shown) and there was no detectable difference with regard to survival. It has been suggested that HDAC inhibitors regulate transcriptional activity through acetylation of histones and chromatin remodeling. In addition, there is increasing evidence that the molecular mechanisms of HDAC inhibition also involve nonhistone acetylation. We sought to investigate the acetylation level of histones in spleens of SAHA-treated animals by Western blot analysis as a marker for in vivo activity of SAHA and HDAC inhibition. For this purpose, mice were treated with SAHA or vehicle and sacrificed on day +6 post-allo-BMT. Splenocytes were prepared and nuclear histones were isolated using acid extraction. Immunoblotting was performed using a polyclonal anti-acetylated H4 antibody. As shown in Figure 1B, in vivo treatment with SAHA resulted in an increased accumulation of acetylated H4 histones in spleen cells on day +6 post-BMT compared to vehicle-treated animals. HDAC inhibitors have been shown to induce apoptosis and cell cycle arrest in tumor cells and cell lines. To rule out that the GVHD-modulating effect of SAHA is mediated through a direct T-cell inhibitory effect, we studied the effect of SAHA on alloantigen-driven T-cell proliferation in vitro in standard MLR assays. Furthermore, SAHA-treated animals were studied with regard to expansion of donor T cells following induction of GVHD. In vitro SAHA was not able to abrogate alloantigen-driven proliferation in a classical one-way MLR up to a concentration of 0.5 μM (Fig. 2A). However, at 1 μM inhibition of T-cell proliferation was observed (Fig. 2A), which was most probably due to induction of cell death by SAHA (data not shown). In line with these in vitro studies we did not observe SAHA-mediated effects on the total number donor CD4+ or CD8+ T cells on days +3 and +7 post-BMT (Fig. 2B) in the spleen of animals following induction of GVHD. Nor did we see an effect on in vivo T-cell activation as determined by the level of CD25 expression on CD4+ and CD8+ T cells (Fig. 2C). Induction of GVHD was associated with a significant upregulation of CD25 on CD8+ T cells on days +3 and +7; this antigen expression was not affected in SAHA-treated animals. To delineate the effect of SAHA on alloantigen and LPS-induced responses we studied the effect of SAHA on alloantigen- or LPS-induced responses in vitro. For this purpose BALB/c splenocytes were stimulated with either irradiated B6 stimulator cells or irradiated syngeneic BALB/c splenocytes with or without LPS and in the presence or absence of SAHA. Supernatants were collected after 24 and 48 hours of stimulation and studied for cytokine or chemokine secretion. As shown in Figures 3A and B the presence of SAHA had significant effects on LPS- and alloantigen-induced cytokine and chemokine production at 48 hours of in vitro stimulation. Thus most notably SAHA completely abolished LPS-induced IFN-γ secretion and also greatly reduced TNF-α, IL-6, and interferon-gamma-inducible protein 10 (IP-10) production. SAHA treatment also significantly affected alloantigen-induced secretion as demonstrated by the inhibition of TNF-α, IL-6, IFN-γ, IL-5, IL-12, IP-10, and macrophage inflammatory protein 1-α. Interestingly, SAHA treatment did not alter IL-10 levels. To further evaluate the effect of SAHA on the early inflammatory response following conditioning and allogeneic BMT, we studied the systemic secretion of chemokines and cytokines after BMT using a cytokine/chemokines multiplex assays. As shown in Figure 4 SAHA-treated allogeneic recipients showed significant decreases in systemic levels of IFN-γ (day +3), TNF-α (day +6), and IL-2 (days +3 and +6), consistent with modulation of the effector function of lymphoid cells. To further assess the influence of SAHA treatment on inflammatory signaling pathways during induction of GVHD, we aimed at dissecting the influence of SAHA treatment on the two main pathogenic factors in the development of GVHD: LPS and alloantigen activation. For analysis of alloantigen-dependent activation splenocytes from B6 and BALB/c mice were cultivated in a classical one-way MLR culture. Furthermore, splenocytes were cultured in the presence or absence of LPS. In the presence of 0.5 μM SAHA we were able to observe a marked inhibition of LPS and alloantigen-induced Tyr701-STAT1 phosphorylation. SAHA treatment resulted in a 50% and 81% inhibition of LPS-induced Tyr701-STAT1 phosphorylation at 4 and 24 hours of incubation, respectively. In contrast SAHA-dependent inhibition (70%) of alloantigen-induced STAT1 phosphorylation became apparent at 24 hours. SAHA treatment, however, was also associated with a downregulation of unphosphorylated STAT1, although less pronounced. Furthermore, STAT1/GAPDH ratio (Fig. 5C) was not significantly affected by SAHA treatment, underscoring the notion that the changes in STAT1 protein levels and STAT1 phosphorylation were not due to SAHA-induced toxicity. In addition to STAT1, as shown in Figure 5D, SAHA treatment also inhibited Tyr-705-STAT3 phosphorylation. Having demonstrated that SAHA treat
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